Nox/Duox enzymes - NADPH-oxidases that generate superoxide and secondary reactive oxygen species (ROS) - participate in normal physiology including cell signaling, innate immunity, thyroid hormone synthesis, and gravity perception. Over-production of ROS by these enzymes is associated with molecular damage and aberrant signaling in disease classes such as hyperproliferative disorders (e.g., cancer, hypertension, atherosclerosis), fibrotic disease (pulmonary fibrosis, cirrhosis, diabetic nephropathy), inflammatory disorders (ARDS, arthritis, atherosclerosis), and reperfusion injury (stroke, myocardial infarction, organ transplantation). The seven human Nox isoenzymes reflect three modes of regulation: 1) constitutively active (Nox4); 2) activation by assembly with regulatory subunits (Nox1, Nox2 and Nox3); and 3) calcium-activated (Nox5, Duox1 and Duox2). We will study the molecular mechanisms of regulation of the catalytic subunits, using Nox2 as representative of subunit-regulated Noxes, Nox4 as a constitutively active Nox, and Nox5 as a Ca2+regulated Nox. The underlying hypothesis to be explored is that all three activation mechanisms induce the same active conformation in the catalytic moiety, allowing electron flow from NADPH to form ROS. Regions on the catalytic subunit involved in responding to calcium or subunits will be identified and characterized, and information will be integrated using a newly developed homology structural model of the Nox catalytic subunit. We will explore the possibility of catalytically essential dimerization, and will investigate the roles of key conserved protein regions identified by an evolutionary comparison of more than 100 Nox enzymes in multiple species. A molecular understanding of the regulation of Nox/Duox enzymes will provide key information that will be key to preventing excess or inappropriate ROS generation and mitigating the course of these diseases. PUBLIC HEALTH RELEVANCE: Nox/Duox enzymes generate a form of free radical referred to as reactive oxygen species (ROS), which is used in normal biological processes to regulate many types of cells and to play a role in the ability of white blood cells to fight infections, the thyroid to produce hormones, and many other normal functions. However, in disease, overproduction of ROS by these enzymes causes both tissue damage and abnormalities in basic cellular functions, and plays a key role in some cancers, complications of diabetes, stroke, cardiovascular diseases, and many other diseases. In order to prevent the progression of these diseases, it is essential to understand the molecular changes that turn on these enzymes to produce too much ROS. This proposal centers on understanding the fundamentals of this process, and has direct implications, for example in our ability to design new classes of drugs that treat these diseases by targeting Nox/Duox enzymes.